The population of Tachypleus tridentatus, a horseshoe crab, has decreased sharply in Japan. With the exception of a few reports regarding the morphological features of T. tridentatus, a three-dimensional analysis of its inner anatomical structures has never been documented. Here, the anatomical structures in two T. tridentatus specimens were examined using magnetic resonance imaging (MRI). In the larger of the two specimens, the three-dimensional T2-weighted image clearly revealed muscles, digestive and urinary systems, ovary, and brain; however, the cardiovascular system was poorly visualized. In the smaller specimen, cardiovascular, digestive, and urinary systems were identifiable. In the three-dimensional T1-weighted images, tissue contrast was low, making it challenging to distinguish the individual organs. These results suggest that MRI is a powerful tool for visualizing the inner anatomical structures of horseshoe crabs.